Neuropeptides are important signaling molecules that regulate brain states, modify neural activity and control vascular tone in the nervous system. Pharmacological and molecular genetic studies have implicated changes in neuropeptide signaling with brain dysfunctions, such as alcohol abuse, drug addiction, and stress. Released via dense-core vesicles into the extrasynaptic space, neuropeptides diffuse over long distances (i.e., volume transmission), and activate G protein coupled neuropeptide receptors. Although widely expressed in the brain, remarkably little is known about their actions on neural circuits. Lacking in the field is a technique to control the timing and spatial release of neuropeptides in real-time in awake animals. To address this unmet need, we will develop a new tool for photo-release of three neuropeptides (SST, VIP, Dyn) using an innovative nano- technology. We will create lipid nano-vesicles (50~100 nm) that encapsulate neuropeptides, and are coated with gold nanoparticles, making them exquisitely sensitive to disruption with two-photon (2p) or one-photon (1p) light. We will integrate the use of photosensitive neuropeptide nano-vesicle with cell-based neurotransmitter fluorescent engineered reporters (CNiFERs), a recently developed technique for optical detection of neuropeptides in vivo. Specifically, we propose to (1) develop photosensitive neuropeptide nano-vesicles (nVs) with efficient and multicolor two-photon release, (2) validate and investigate modulation of photo-released neuropeptide in brain slices, and (3) determine the properties of photo-released neuropeptides in vivo and the impact on neuronal excitability in the cerebral cortex. With the all-optical release and monitoring, we will investigate the diffusion rate and distance for neuropeptides in vivo. We provide preliminary data demonstrating feasibility of creating and studying photo-released peptides. Completion of the proposed research will establish a transformative technique to investigate neuropeptide volume transmission and its modulation of neural circuits. Success of this proposed research will advance studies on the transient and localized effects of neuropeptides that are currently not possible with today's technology, revealing new information on the function of neuropeptides in the brain.